Isolation of a mRNA for the 18,000 dalton maize heat shock polypeptide

Plants respond to heat shock by synthesizing a set of heat shock polypeptides (HSPs) (C. L. Baszczynski et al., Can. J. Bioch. 60:569, 1982, P, Cooper et al., Plant Phys. 75:431, 1984). Poly A + RNA isolated from 5-6 day old seedlings following heat shock directs the synthesis of the HSPs, in both a rabbit reticuloycte lysate system and a wheat germ system (Baszczynski et al., Can. J. Bioch. 61:395, 1983). RNA was isolated as previously described from plumules of 5-6 day old seedlings (Oh43) following temperature shift (25 C-42 C, 1 h). Oligo-dT-chromatography of the RNA was carried out and the poly A + fraction was collected. Approximately 2 mg/g fresh tissue of RNA were recovered, of which 20% was the poly A + fraction. This fraction was contaminated with rRNA (seen as 28S and 18S bands on agarose gels) even after repeated passage through the column.

Agarose gel electrophoresis (under denaturing conditions) was used to separate RNA molecules. Use was made of mRNA affinity paper, which reversibly binds poly A+ RNA (D. H. Wreschner and M. Hersberg, Nucl. Acid. Res. 12:1351, 1984). Poly A+ RNA was separated in urea/citrate denaturing, 1.75% agarose gels (R. E. Smith and Y. Furuichi, Virology 103:279, 1980). This "non-invasive" denaturing system was recommended by the developers of mAP paper. RNA samples (50 µg/3.5 cm well) were prepared by diluting with an equal volume of 9 M urea, boiling for 1 minute and cooling on ice. Gel electrophoresis was carried out for 22 h (2 V/cm, RT). Standard and sample lanes for staining were cut from the gel and stained with ethidium bromide (2 µg/ml). Sample lanes were equilibrated in 500 mM Tris HCl pH 7.6 for 2 h. The gel was blotted to mAP paper using the same buffer (20 h). The mAP paper was washed in transfer buffer and sections containing mRNA (determined from the stained sample lanes) were cut from each lane. The sections were cut sequentially into 0.5 cm pieces which were rinsed in ethanol (70%), dried and placed in 0.6 ml Eppendorf tubes. Sterile ddH2O (100 µl) was added and the tubes were placed at 70 C for 3 minutes. The tubes were punctured at the bottom and placed in 1.5 ml Eppendorf tubes which were centrifuged 3 min. The RNA was ethanol precipitated, dried briefly and translated in a rabbit reticulocyte lysate system using 35S methionine as labeled precursor. Translation products were separated by SDS-PAGE and fluorography was conducted. Examination of the fluorograms showed a prominent translation product of molecular mass 18,000 daltons. The message which translates into this polypeptide was eluted from an agarose gel slice determined to contain RNA molecules 630-850 nucleotides in length. Work is underway to prepare complementary DNA to this RNA using reverse transcriptase.

C. A. Rees


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